[박상규] Adipogenesis of p43 MEF

[Anonymous]

Adipogenesis of p43 MEF (E14.5 day)

 

 

 

1.     Culture p43 MEF cells in DMEM containing 10% FBS and 1% antibiotics till confluent

2.     Exchange with fresh media and culture for 2 days more.

3.     Exchange with fresh media containing MDI

4.     Repeat step 3 every day (24 hrs interval) till day 8-13

5.     Fix the cells with 5% formalin (in PBS) for 20 min at room temperature

6.     Rinse cells with PBS 3 times

7.     Stain with 0.3 % Oil Red O solution for 1 hr at room temperature

8.     Rinse with water for at least 30 min to remove the residual junk of Oil Red O

9.     In the case of dish, store at DW until visualization under the microscope.

 

 

MDI

M: Methylisobutylxanthine (inhibit proliferation)è stock : 1M in DMSO=> working conc : 500uM

D: Dexamethasone (induce differentiation)è stock:2mM in ethanol=> working conc : 1-2uM

I: Insulin (induce differentiation)è stock: 10mg/ml => working conc : 10ug-20ug/ml

 

If you purchase the insulin powder, you should be very careful to make stock

solution because the insulin activity is sensitive to pH. So I recommend you had

better purchase the pre-made solution.

 

In most case, if you treat MDI sol for 8 day, you can see the differentiation pattern

of MEF. However, in some cases, you have to treat the MDI for 14 days to see the

adipocyte differentiation

 

You have to culture MEF within passage 7. If you culture MEF cells over passage 7,

Differentiation may not be induced by MDI sol.

[Author]

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